Identification of gene sequences and proteins involved in vaccinia virus dominant T cell epitopes

ABSTRACT

The present invention relates to the identification of gene sequences and proteins involved in vaccinia virus dominant T cell epitopes. Two vaccinia virus CD8 +  T cell epitopes restricted by the most common human MHC class I allele, HLA-A0201 have been identified. Both epitopes are highly conserved in vaccinia and variola viruses. The induction of the T cell responses following primary vaccination is demonstrated by the kinetics of epitope specific CD8 +  T cells in 3 HLA-A0201 individuals. This information will be useful for the design and analyses of the immunogenicity of experimental vaccinia vaccines, and for basic studies of human T cell memory.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/442,846, filed Jan. 24, 2003. The entire teachings of the aboveapplication is incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant PO1AI-49320 and a subcontract, AI-46725 from the National Institutes ofHealth/National Institute of Allergy and Infectious Diseases. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Immunization with vaccinia virus resulted in long-lasting protectionagainst smallpox and was the successful approach used to eliminatenatural smallpox infections worldwide. This accomplishment was achievedwithout a detailed understanding of human T cell responses topoxviruses. Due to the concern about the potential use of smallpox virusas a bioweapon, smallpox vaccination is currently being reintroduced.However, severe and life threatening complications from vaccination wereassociated with congenital or acquired T cell deficiencies, but not withcongenital agammaglobulinemia. Considering the high incidence of sideeffects from current smallpox vaccine, the development of a safer, butequally effective vaccine is very important. Thus, it is important tohave a detailed understanding of human T cell responses to poxviruses.

Vaccinia-specific CD4⁺ and CD8⁺ T cells have been detected in humans andthe number of vaccinia virus-specific T cell responses to smallpoxvaccine have been measured. Additionally, an intracellular cytokinestaining assay was applied to quantitate and characterizevaccinia-specific T cells in mice. However, no T cell epitopes have beenidentified in humans or mice systems. One major obstacle is the size ofthe virus. Vaccinia is a large virus with an approximately 200-kbp DNAgenome that has approximately 200 open reading frames.

In order to analyze T cell responses to licensed and experimentalsmallpox vaccines at the single cell level, it is essential to identifyCD8⁺ T cell epitopes. In addition to emphasizing the importance of Tcells in the immunity to smallpox, there is a critical need to developnew vaccines safe for use in T cell deficient populations. Thisinformation will be useful for the design and analyses of theimmunogenicity of experimental vaccinia vaccines, and for basic studiesof human T cell memory.

SUMMARY OF THE INVENTION

The present invention relates to the identification of gene sequencesand proteins involved in vaccinia virus dominant T cell epitopes. In oneembodiment, the invention provides a method for immunizing an individualagainst infection by vaccinia and/or variola virus, the methodcomprising inducing an immune response against a polypeptide comprisingpeptide 74A. In this embodiment, the polypeptide can be selected fromthe group consisting of MVA189R, Copenhagen B22R, Copenhagen C16L,Bangladesh-1975 D2L, India-1967 D1L, Garcia-1966 B1L, Brighton Red V212or Zaire-96-I-16 N1R or other homologues of vaccinia and variola virus.In another embodiment, the method can further comprise a secondpolypeptide comprising peptide 165. In a further embodiment, the immuneresponse is induced by administering a product selected from the groupconsisting of a polypeptide, a naked nucleic acid molecule encoding thepeptide or a nucleic acid molecule, encoding the peptide, in a suitablevector.

In another embodiment, the present invention relates to a method forimmunizing an individual against infection by vaccinia and/or variolavirus, the method comprising inducing an immune response against apolypeptide comprising peptide 165. In this embodiment, the polypeptidecan be selected from the group consisting of MVA018L, Copenhagen C7L,Tian Tan TC7L, Bangladesh-1975 D11L, India-1967 D8L, Garcia-1966 B14L,Brighton Red V028 or Zaire-96-I-16 D10L or other homologues of vacciniaand variola virus. In a further embodiment, the method can furthercomprise a second polypeptide comprising peptide 74A. In a furtherembodiment, the immune response is induced by administering a productselected from the group consisting of a polypeptide, a naked nucleicacid molecule encoding the peptide or a nucleic acid molecule, encodingthe peptide, in a suitable vector.

In another embodiment, the present invention relates to a method forimmunizing an individual against infection by vaccinia and/or variolavirus, the method comprising inducing an immune response against apolypeptide comprising peptide 74A, immunogenic fragments or mutantsthereof. From 1 to about 4 amino acids can be substituted to make up theimmunogenic fragments or mutants of peptide 74A, without essentiallydetracting from the immunological properties of peptide 74A. In thisembodiment, the polypeptide can be selected from the group consisting ofMVA189R, Copenhagen B22R, Copenhagen C16L, Bangladesh-1975 D2L,India-1967 D1L, Garcia-1966 B1L, Brighton Red V212 or Zaire-96-I-16 N1Ror other homologues of vaccinia and variola virus. In anotherembodiment, the method can further comprise a second polypeptidecomprising peptide 165, immunogenic fragments or mutants thereof. In afurther embodiment, the immune response is induced by administering aproduct selected from the group consisting of a polypeptide, a nakednucleic acid molecule encoding the peptide or a nucleic acid molecule,encoding the peptide, in a suitable vector.

In another embodiment, the present invention relates to a method forimmunizing an individual against infection by vaccinia and/or variolavirus, the method comprising inducing an immune response against apolypeptide comprising peptide 165, immunogenic fragments or mutantsthereof. From 1 to about 4 amino acids can be substituted to make up theimmunogenic fragments or mutants of peptide 165, without essentiallydetracting from the immunological properties of peptide 165. In thisembodiment, the polypeptide can be selected from the group consisting ofMVA018L, Copenhagen C7L, Tian Tan TC7L, Bangladesh-1975 D11L, India-1967D8L, Garcia-1966 B14L, Brighton Red V028 or Zaire-96-I-16 D10L or otherhomologues of vaccinia and variola virus. In another embodiment, themethod can further comprise a second polypeptide comprising peptide 74A,immunogenic fragments or mutants thereof. In a further embodiment, theimmune response is induced by administering a product selected from thegroup consisting of a polypeptide, a naked nucleic acid moleculeencoding the peptide or a nucleic acid molecule, encoding the peptide,in a suitable vector.

The present invention also relates to a method of identifying thepresence of vaccinia, variola or other related poxvirus in a samplecomprising determining whether T cells present in the sample (e.g.,blood, lymph and tissue) become activated in the presence of apolypeptide selected from the group consisting of: peptide 74A (SEQ IDNO: 1), peptide 165 (SEQ ID NO: 2) and a combination thereof, wherein ifthe T cells become activated, then vaccinia, variola or other relatedpoxvirus is present in the sample. Whether the T cells present in thesample become activated can be determined using, for example, a cytokineassay (e.g., ELISPOT), a flow cytometry assay (e.g., tetramer stainingassay)and/or a limiting dilution assay.

T cells can be present in the original sample or can be added to thesample. For example, the sample can be blood which contains T cells. Inthis embodiment, whether the T cells become activated in the presence ofthe polypeptide is determined, wherein if the T cells become activated,then vaccinia, variola or other related poxvirus is present in thesample. In another embodiment, the sample does not initially contain Tcells. In this embodiment, the sample is contacted with T cells thatbecome activated in the presence of a vaccinia, variola or other relatedpoxvirus. Then whether the T cells become activated in the presence ofthe polypeptide is determined, wherein if the T cells become activated,then vaccinia, variola and/or other related poxvirus is present in thesample.

The present invention also relates to a method of determining whether anindividual has been infected with vaccinia, variola or other relatedpoxvirus comprising determining whether the individual's T cells becomeactivated in the presence of polypeptide selected from the groupconsisting of: peptide 74A (SEQ ID NO: 1), peptide 165 (SEQ ID NO: 2)and a combination thereof, and wherein if the individual's T cellsbecome activated in the presence of the peptide, then the individual hasbeen infected with vaccinia, variola or other related poxvirus.

A method of monitoring the effectiveness of a vaccinia, variola or otherrelated poxvirus vaccine in an individual who has been administered thevaccinia vaccine is also encompassed by the present invention. Themethod comprises determining whether the individual's T cells becomeactivated in the presence of a polypeptide selected from the groupconsisting of: peptide 74A (SEQ ID NO: 1), peptide 165 (SEQ ID NO: 2)and a combination thereof, wherein if the individual's T cells becomeactivated, then the vaccine is effective in the individual. In oneembodiment, the vaccine is a vaccinia virus vaccine. In anotherembodiment, the vaccinia virus vaccine is a cancer vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a schematic depicting the kinetics of the frequency ofIFN-γ-producing cells specific to these peptides and to whole vacciniavirus quantitated by IFN-γELISPOT assay using the peripheral bloodmononuclear cells (PBMCs) of the vaccinia-immune donors. Closed circle:peptide 74A-specific cells. Open circle: peptide 165-specific cells.Closed square: vaccinia virus-specific cells. P: pre-immunization.

FIG. 2 depicts FACS staining to confirm specificity of MVA74A and MVA165tetramers. HLA-A0201-negative donor PBMC was mixed with CD8⁺ T cellclones specific for either the MVA74A or the MVA165 vaccinia virusepitope at a ratio of 10:1. Four color FACS analysis was done todetermine the specificity. Cells are gated on CD3⁺ CD4⁻ cells withtetramer on the X-axis and CD8 on the y-axis.

FIG. 3 is a schematic depicting the quantitation of vaccinia virusepitope-specific CD8⁺ T cells by HLA-A0201/peptide 74A tetramer staining(top) and HLA-A0201/peptide 165 tetramer staining (bottom) of PBMCs ofdonor 1. Cells were gated on CD3⁺ and CD4⁻ cells with tetramer stainingon the x-axis and CD8 on the y-axis. The larger squares show CD8⁺ cellsand the smaller squares show CD8⁺ and tetramer⁺ cells.

FIG. 4 is a schematic depicting the kinetics of the frequency of CD8⁺ Tcells specific for each epitope quantitated by tetramer staining inPBMCs of three donors after primary immunization. Closed circle:74A-specific. Open circle: 165-specific. P: pre-immunization. Frequencyis calculated per million PBMC for comparison with the data fromepitope-specific IFN-γ ELISPOT assays.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

Successful vaccines deliver to a host one or more antigens derived froma pathogen, thereby stimulating an immune response which protectsagainst subsequent challenge with the pathogen. Such vaccines can take avariety of forms, including attenuated or killed pathogens, for example,viruses or bacteria; one or more proteins or peptides derived from apathogen or synthetic or recombinant versions of such proteins orpeptides; or one or more nucleic acid molecules encoding one or moreproteins or peptides from the pathogen, such as a naked DNA vaccine or anucleic acid molecule administered in a suitable vector, such as arecombinant virus or bacterium or an immunostimulating complex. Vaccinesagainst cell proliferative diseases, such as cancers, typically utilizeproteins or fragments thereof, or nucleic acid molecules encodingproteins or fragments thereof, which are unique to diseased cells orgenerally more abundant in diseased cells compared to healthy cells.

Cell-mediated immunity is dependent upon lymphocytes known as B cellsand T cells. B cells produce antibodies targeted against extracellularantigens. T cells recognize antigen fragments (peptides) which aredisplayed at the surface of a host cell. Such antigen fragments resultfrom uptake of the antigen by a host cell, or synthesis of the antigenwithin the host cell, followed by cleavage of the antigen within thecell.

Foreign proteins which are synthesized within the host cell or are takenup by the host cell via specific receptors are fragmented within thecytosol of the cell. One or more of the resulting peptides can becomeassociated with class I major histocompatibility molecules (MHC I), andthe resulting complexes are then presented at the surface of the cell.These MHC I/peptide complexes are recognized by specific T cellreceptors in certain CD8⁺ T cells, and the peptides so presented arereferred to as CD8 epitopes.

A foreign protein can be taken up by a host cell nonspecifically viaendocytosis and then fragmented into peptides in a cellular lysosomal orendosomal compartment. One or more of these peptides can then becomeassociated with a class II major histocompatibility molecule (MHC II) toform a complex which is then presented at the surface of the host cell.These MHC II/peptide complexes are recognized by CD4⁺ T cells expressinga specific receptor which recognizes the MHC II/peptide complex. Thesepeptides are referred to as CD4 epitopes.

Peripheral T cells in the blood and organs of the immune system (e.g.spleen and lymph nodes) exist in a quiescent or resting state. Uponinteraction of T cells with an MHC/epitope complex, the T cellsproliferate and differentiate into activated cells having a variety offunctions. CD8⁺ T cells typically become cytotoxic upon activation anddestroy antigen-presenting cells via direct contact. Activated CD4⁺ Tcells provide a helper function to B cells, enabling B cells todifferentiate into antibody-producing cells. Activated CD8⁺ T cells andCD4⁺ T cells release a variety of cytokines (lymphokines orinterleukins), which can, for example, control differentiation of manyclasses of lympholytic precursor cells.

The present invention relates to vaccinia virus-specific CD8⁺ cytotoxicT lymphocyte (CTL) lines that were established by limiting dilutioncloning from the peripheral blood mononuclear cells (PBMCs) ofHLA-A0201-positive donors who received primary immunization with thelicensed smallpox vaccine, Dryvax®. Among the highly polymorphic humanMHC class I genes, HLA-A0201 was chosen to identify CD8⁺ T cell epitopesbecause of the commonality of this allele among most ethnic groups.HLA-A0201 peptide binding motif searches was performed on all of theprotein sequences of the modified vaccinia virus Ankara (MVA) strain(GenBank accession number U94848), which is being proposed for use as anattenuated smallpox vaccine and as a vector for vaccination againstother infectious agents. The computer algorithm “HLA Peptide BindingPredictions” (on the World Wide Web at:bimas.dcrt.nih.gov/molbio/hla_bind/ visited on Aug. 20, 2001 and Aug.21, 2001) was used to calculate the binding affinity (score) of 9 merpeptides to the HLA-A0201 molecule. It was hypothesized that early geneproducts may be more likely to have CD8⁺ T cell epitopes, since in bothhumans and mice all of the known CD8⁺ T cell epitopes to cytomegalovirusare encoded by immediate-early phase proteins. The early, early andlate, and late genes in vaccinia were categorized by nucleotide sequencemotifs, such as a late promoter or an early termination motif. Forinitial screening all peptides with; (1) a binding score of more than a1000 (70 peptides); or (2) a binding score of 100 to 999 and encoded bya gene expressed early or both early and late (125 peptides) weresynthesized. A total of 195 peptides were screened using fifteenvaccinia virus-specific CTL lines. Two T cell epitopes were restrictedby HLA-A0201 and cross-reactive to MVA.

One CTL line, VA55 3.13, recognized peptide 74A, CLTEYILWV (SEQ ID NO:1), in a 21.7K protein encoded by a putative early and late gene,“189R”, of the MVA strain with a calculated binding score of 3607.Another CTL line, VA49 3.12, recognized peptide 165, KVDDTFYYV (SEQ IDNO: 2), which is in a host range protein encoded by a putative early andlate gene, “018L”, with a calculated binding score of 365. FIG. 1demonstrates the high level of specific recognition by these CTL linesof their respective epitope peptides (i.e., peptide 74A or peptide 165)in a dose response CTL experiment. These epitope sequences arehighly-conserved in vaccinia and variola viruses (Table 1).

TABLE 1 Conservation of epitopes among poxviruses causing infection inhuman Only strains of which complete genome has been sequenced arelisted. GenBank Gene accession # Gene name 74A peptide name 165 peptideVaccinia MVA U94848 189R CLTEYILWV 018L KVDDTFYYV (SEQ ID (SEQ ID NO: 1)NO: 2) Copenhagen M35027 B22R & C16L^(b) ********* C7L ********* TianTan^(a) AF095689 TC7L ********* Variola major Bangladesh-1975 L22579 D2L********* D11L ********* India-1967 X69198 D1L ********* D8L *********Variola minor Garcia-1966 Y16780 B1L ********* B14L ********* CowpoxBrighton Red AF482758 V212 ********* V028 ********* MonkeypoxZaire-96-I-16 AF380138 N1R ********* D10L ***Y*L*** (SEQ ID NO: 3) *indicate identical amino acid. ^(a)Tian Tan strain does not have 189Rorthologue according to the nucleotide sequence. ^(b)Both genes arelocated within the inverted terminal repeats and the gene sequences areidentical.

Epitope-specific T cell clones can be generated using methods which aregenerally known in the art (see, for example, Fathman, et al., in Paul,ed., Fundamental Immunology, second edition, Raven Press (1989), Chapter30, the contents of which are hereby incorporated by reference in theirentirety). The isolation of epitope-specific T cell clones is based on Tcell biology. Generally, an animal, such as a mouse, is immunized with apreparation of antigens (a bacterial lysate, or a purified protein) oris infected with a virus, such as a wild type virus or a recombinantvirus containing heterologous genes encoding one or more proteins from apathogenic microorganism, such as a virus. The animal is then sacrificedand the peripheral blood mononuclear cells (PBMC: includes T cells, Bcells, monocytes), spleen and lymph nodes are isolated. The isolatedcells are then cultured in media containing a defined component of theoriginal antigenic preparation, often a recombinant or purified protein,and the essential T cell growth factor interleukin-2 (IL-2). The only Tcells which will proliferate are those which recognize MHC/epitopecomplex in which the epitope is derived from the antigenic preparation.These cells become activated and proliferate while the unactivated cellsbegin to die. The cultures are maintained for several weeks, with themedia containing antigen and IL-2 being periodically replaced.Eventually, clusters of living and dividing cells (a T cell line) can beobserved in some of the cultures.

The proliferating cells are generally not clonal at this point and areof limited use for assaying epitope specific T cell responses. The Tcell line is, preferably, cloned through a process referred to aslimiting dilution. In this method, PBMC are isolated from, for example,the same strain as the original used to isolate the T cell line. Thesecells, called antigen presenting cells, will serve as a source of MHCproteins and will present the MHC:peptide complex to the T cell line.The T cell line is diluted to a concentration of about 1 to 5 T cells/mLin a suspension of APCs that contains the antigen of interest and IL-2.This suspension is then transferred into, for example, round or“v”-bottom 96 well microtitre plates, so that each well contains, onaverage, no more than 1 T cell. The cultures are maintained for severalweeks and a clone can grow out of one or more cultures. The cellsisolated by limiting dilution are the progeny of a single cell thatexpresses only one T cell receptor, and the clone is thusepitope-specific.

CD8⁺ T cells specific to these epitopes were measured at several timepoints following primary immunization by peptide/HLA-A0201 tetramerstaining using the PBMCs of three HLA-A0201-positive donors. FIG. 2shows representative FACS plots of donor 1 PBMC. In FIGS. 3 and 4,“preimmune” means prior to primary first immunization, and “two weeks”means two weeks after the second immunization for donor 3 who failed to“take” after primary immunization, and twenty days after primaryimmunization was immunized for the second time. In all three donors thefrequency of vaccinia-specific CD8⁺ T cells peaked two weeks afterprimary immunization and then declined, but were still detectable one tothree years following primary immunization (FIG. 3). Two weeks aftervaccination the IFN-γ-producing cells specific to these two epitopeswere 14% of total vaccinia virus-specific IFN-γ-producing cells in donor1, 35% in donor 2, and 6% in donor 3 (FIG. 4).

Thus, two CD8⁺ T cell epitopes restricted by HLA-A0201, the most commonMHC class I allele in humans have been identified. These are the first Tcell epitopes that have been reported for vaccinia virus.IFN-γ-producing cells specific to these two epitopes represented 6 to35% of total number of IFN-γ-producing cells specific to vaccinia virus.The frequency of epitope-specific T cells was always higher bypeptide/HLA tetramer staining than by IFN-γ-ELISPOT assay, althoughpost-vaccination kinetics for each epitope-specific T cell was similarusing both methods.

As for epitope selection, peptide 74A was the 15^(th) highest bindingpeptide to HLA-A0201 of the 195 peptides selected for screening andpeptide 165 was the 95^(th) highest binder. One common characteristic ofthese two peptides is that they are both encoded by genes with a latepromoter and an early termination motif, which means they may beexpressed at both early and late phases of infection. The 189R gene ofMVA strain encoding peptide 74A is a nonessential gene with unknownfunction. The 018L gene of MVA encoding peptide 165 is an orthologue ofthe host range protein, C7L, of the Copenhagen strain. Althoughselection of peptides was biased toward genes expressed in the earlyphase of infection, viral proteins produced in the early phase ofinfection may be processed and presented more efficiently by infectedcells than those produced only in late phase, as a result of vacciniavirus down regulating host protein synthesis. These two epitopes arehighly-conserved among variola viruses, suggesting the CTLs recognizingthese epitopes will recognize variola virus-infected cells.

In one embodiment, the invention provides a method for immunizing anindividual against infection by vaccinia and/or variola virus, themethod comprising inducing an immune response against a polypeptidecomprising peptide 74A. In this embodiment, the polypeptide can beselected from the group consisting of MVA189R, Copenhagen B22R,Copenhagen C16L, Bangladesh-1975 D2L, India-1967 D1L, Garcia-1966 B1L,Brighton Red V212 or Zaire-96-I-16 N1R or other homologues of vacciniaand variola virus. In another embodiment, the method can furthercomprise a second polypeptide comprising peptide 165. In a furtherembodiment, the immune response is induced by administering a productselected from the group consisting of a polypeptide, a naked nucleicacid molecule encoding the peptide or a nucleic acid molecule, encodingthe peptide, in a suitable vector.

In another embodiment, the present invention relates to a method forimmunizing an individual against infection by vaccinia and/or variolavirus, the method comprising inducing an immune response against apolypeptide comprising peptide 165. In this embodiment, the polypeptidecan be selected from the group consisting of MVA018L, Copenhagen C7L,Tian Tan TC7L, Bangladesh-1975 D11L, India-1967 D8L, Garcia-1966 B14L,Brighton Red V028 or Zaire-96-I-16 D10L or other homologues of vacciniaand variola virus. In a further embodiment, the method can furthercomprise a second polypeptide comprising peptide 74A. In a furtherembodiment, the immune response is induced by administering a productselected from the group consisting of a polypeptide, a naked nucleicacid molecule encoding the peptide or a nucleic acid molecule, encodingthe peptide, in a suitable vector.

In another embodiment, the present invention relates to a method forimmunizing an individual against infection by vaccinia and/or variolavirus, the method comprising inducing an immune response against apolypeptide comprising peptide 74A, immunogenic fragments or mutantsthereof. From 1 to about 4 amino acids can be substituted to make up theimmunogenic fragments or mutants of peptide 74A, without essentiallydetracting from the immunological properties of peptide 74A. In thisembodiment, the polypeptide can be selected from the group consisting ofMVA189R, Copenhagen B22R, Copenhagen C16L, Bangladesh-1975 D2L,India-1967 D1L, Garcia-1966 B1L, Brighton Red V212 or Zaire-96-I-16 N1Ror other homologues of vaccinia and variola virus. In anotherembodiment, the method can further comprise a second polypeptidecomprising peptide 165, immunogenic fragments or mutants thereof. In afurther embodiment, the immune response is induced by administering aproduct selected from the group consisting of a polypeptide, a nakednucleic acid molecule encoding the peptide or a nucleic acid molecule,encoding the peptide, in a suitable vector.

In another embodiment, the present invention relates to a method forimmunizing an individual against infection by vaccinia and/or variolavirus, the method comprising inducing an immune response against apolypeptide comprising peptide 165, immunogenic fragments or mutantsthereof. From 1 to about 4 amino acids can be substituted to make up theimmunogenic fragments or mutants of peptide 165, without essentiallydetracting from the immunological properties of peptide 165. In thisembodiment, the polypeptide can be selected from the group consisting ofMVA189R, Copenhagen C7L, Tian Tan TC7L, Bangladesh-1975 D1L, India-1967D8L, Garcia-1966 B14L, Brighton Red V028 or Zaire-96-I-16 D10L or otherhomologues of vaccinia and variola virus. In another embodiment, themethod can further comprise a second polypeptide comprising peptide 74A,immunogenic fragments or mutants thereof. In a further embodiment, theimmune response is induced by administering a product selected from thegroup consisting of a polypeptide, a naked nucleic acid moleculeencoding the peptide or a nucleic acid molecule, encoding the peptide,in a suitable vector.

Several methods are described in the literature which are useful for theidentification of T cell epitopes. For example, DeLisi et al. havesuggested that potential epitopic sites may be located by identificationof potential amphipathic alpha helical regions in the molecule. DeLisiet al., Proc. Natl. Acad. Sci. USA 82:7048 (1987). Bixler et al.describe a strategy of synthesizing overlapping synthetic peptidesencompassing an entire protein molecule for delineation of T cellepitopes. Bixler et al., Immunol. Com. 12:593 (1983); Bixler et al. J.Immunogenet. 11:339 (1994). A synthetic method described by Gysen (CibaFoundation Symposium 119:130 (1986)) permits synthesis of a largevariety of peptides thereby mimicking of a variety of potential bindingsites, in turn allowing rapid scanning of a molecule.

More traditional methods, such as enzymatic or chemical digestion ofproteins provide peptide fragments which may be tested for T cellactivity. For example, enzymes such as chymotrypsin, elastase, ficin,papain, pepsin, or trypsin provide limited and predictable fragments bycleavage of specified amino acid linkages; similarly chemical compoundssuch as N-chloro-succinimide BPNS-skatole, cyanogen bromide, formicacid, or hydroxylamine, also produce definable fragments by their actionon proteins. The presence of the desired T cell stimulating activity inany given fragment can be determined by subjecting purified fragments toa standard T cell proliferation assay, or by analyzing unpurifiedfragments with a T cell Western Assay. Young et al., Immunol. 59:167(1986).

Peptide 74A and peptide 165 of the invention are CD8 epitopes and the Tcells specific for these peptides are CD8⁺ T cells. The effectorfunctions of CD8⁺ T cells include lysis of antigen presenting cells andrelease of cytokines. Therefore, the extent of CD8⁺ T cell response tothe antigen presenting cells can be determined using an assay for celllysis or by measuring the production of one or more cytokines. The CD8⁺T cell response can also be measured by measuring the extent of releaseof one or more cytokines. In general, greater cell lysis activity orcytokine release will correlate with greater immunogenicity.

In one embodiment, the present invention relates to methods forimmunizing an individual, particularly a human, against infection byvaccinia and/or variola virus by inducing an immune response against apolypeptide comprising peptide 74A and/or peptide 165. In a furtherembodiment, the immune response can be induced against a polypeptidecomprising an immunogenic fragment or mutant of peptide 74A and/orpeptide 165. Although the methods described herein are particularlyuseful for human immunization, the methods are equally applicable toother mammals. In particular embodiments, the individual is positive forthe HLA-A0201 gene.

As used herein the terms “immunogenic fragment” and “mutant” of peptide74A and/or peptide 165 refer to polypeptides in which 1 to about 4 aminoacids have been substituted without essentially detracting from theimmunological properties thereof can be generated in a variety of ways.For example, in vitro mutagenic techniques can be used to modify thecloned gene encoding peptide 74A and/or peptide 165. Such methods, whichare well known to one skilled in the art, can be used to delete, insertor substitute nucleotides in the gene resulting in the deletion,insertion or substitution of amino acids in the encoded product.Examples of immunogenic fragments or mutants of peptide 74A and peptide165 include, but are not limited to, those shown in Table 2. Theimmunological properties of the mutagenized encoded product can beassayed using methods such as those which are well known to one skilledin the art.

TABLE 2 Examples of immunogenic fragments and mutants of peptide 74A andpeptide 165. Possible Immunogenic fragments or mutants 74A peptide 165peptide 1 ILTEYILWV IVDDTFYYV (SEQ ID NO: 4) (SEQ ID NO: 19) 2 LLTEYILWVLVDDTFYYV (SEQ ID NO: 5) (SEQ ID NO: 20) 3 FLTEYILWV FVDDTFYYV (SEQ IDNO: 6) (SEQ ID NO: 21) 4 CLAEYILWV KVADRFYYV (SEQ ID NO: 7) (SEQ ID NO:22) 5 CLYEYILWV KVYDTFYYV (SEQ ID NO: 8) (SEQ ID NO: 23) 6 CLEFEYILWVKVFDTFYYV (SEQ ID NO: 9) (SEQ ID NO: 24) 7 CLTEIILWV KVDDIFYYV (SEQ IDNO: 10) (SEQ ID NO: 25) 8 CLTEKILWV KVDDKFYYV (SEQ ID NO: 11) (SEQ IDNO: 26) 9 CLTENILWV KVDDYFYYV (SEQ ID NO: 12) (SEQ ID NO: 27) 10CLTEYIAWV KVDDTFAYV (SEQ ID NO: 13) (SEQ ID NO: 28) 11 CLTEYIYWVKVDDTFHYV (SEQ ID NO: 14) (SEQ ID NO: 29) 12 CLTEYIHWV IVADTFYYV (SEQ IDNO: 15) (SEQ ID NO: 30) 13 ILAEYILWV IVADIFYYV (SEQ ID NO: 16) (SEQ IDNO: 31) 14 ILAEIILWV IVADIFAYV (SEQ ID NO: 17) (SEQ ID NO: 32) 15ILAEIIAWV LVYDKFHYV (SEQ ID NO: 18) (SEQ ID NO: 33)

Effective dosages for inducing an immune response (also referred to as avirus protective response) against vaccinia and/or variola can bedetermined empirically with initial dosage ranges based upon historicaldata for peptide/protein vaccine compositions. As used herein, the terms“induced immune response” or “virus protective response” refers to animmunological response in the individual resulting in the successfulcontrol or limitation of infection by vaccinia and/or variola viruswhich is clinically observed.

For example, individuals can be administered dosages of peptide 74Aand/or peptide 165 ranging from 0.5–500 micrograms. Whether a particulardosage is effective can be determined using well known T cellproliferation and cytotoxicity assays. For example, followingadministration of the protein to an individual blood is drawn. CytotoxicT cells are identifiable by ⁵¹Cr release assay (see e.g., Kuwano et al.,J. Virol. 140:1264–1268 (1988)). Helper T cells are identifiable by astandard T cell proliferation assay (see e.g., Kurane et al., J. Clin.Invest. 83:506–513 (1989)). The results from these studies are comparedwith results from the same experiments conducted with T cells from thesame individual prior to administration of the antigen. By comparingthis data, effective dosage ranges can be determined.

A wide variety of pharmaceutically acceptable carriers are useful.Pharmaceutically acceptable carriers include, for example, water,physiological saline, ethanol polyols (e.g., glycerol or administrationis typically parenteral (i.e., intravenous, intramuscular,intraperitoneal or subcutaneous). An adjuvant (e.g., alum) can also beincluded in the vaccine mixture.

The invention also pertains to a method for immunizing an individualagainst infection by vaccinia and/or variola virus by administering avaccine composition comprising at least one essentially pure T cellepitope (i.e., peptide 74A or peptide 165) in combination with apharmaceutically acceptable carrier. Due to genetic variability betweenindividuals, a single T cell epitope may not stimulate a virusprotective response in all individuals to whom it is administered.Therefore, by combining two or more distinct T cell epitopes (i.e., bothpeptide 74A and peptide 165), the vaccine is more broadly effective. Asindicated above, helper T cell epitopes and cytotoxic T cell epitopesare thought to comprise distinct (albeit possibly overlapping) regionsof proteins. Cytotoxic T cell epitopes can be distinguished from helperT cells epitopes experimentally using the cytoxicity and proliferationassays described above (helper T cells stimulate proliferation but donot posses cytotoxic activity).

Peptide 74A and/or peptide 165 can be administered as an polypeptide.Such polypeptides can be synthesized chemically. Alternatively, atruncated portion of a gene encoding peptide 74A and/or peptide 165 canbe expressed in a cell, and the encoded product can be isolated usingknow methods (e.g., column chromatography, gel electrophoresis, etc.).

As used herein, the term polypeptide means any amino acid sequence whichis identical or substantially homologous to peptide 74A and/or peptide165. The expression substantially homologous refers to polypeptideshaving an amino acid sequence of peptide 74A or peptide 165 in whichamino acids have been substituted without essentially detracting fromthe immunological properties thereof. This definition includes aminoacid sequences of sufficient length to be classified as oligopeptides(these terms are not used consistently or with great precision in theliterature).

In one embodiment, both a helper T cell epitope and a cytotoxic T cellepitope can be administered to the individual. The stimulation ofcytotoxic T cells is desirable in that these cells will kill cellsinfected by vaccinia and/or variola virus. The stimulation of helper Tcells is beneficial in that they secrete soluble factors which have astimulatory effect on other T cells, as well as B cells.

In another embodiment, a gene encoding a protein listed in Table 1, or aportion thereof which contains peptide 74A or peptide 165, can be clonedinto a recombinant virus which expresses peptide 74A or peptide 165, orimmunogenic fragment or mutant thereof, in the individual to beimmunized. An example of such a recombinant virus system is the vacciniasystem described by Paoletti et al. (U.S. Pat. No. 4,603,112), thedisclosure of which is incorporated herein by reference. Other viruseshave been described in the literature which have a genome which canaccommodate the insertion of a foreign DNA such that a protein encodedby the DNA is expressed in vivo. Any such recombinant virus is usefulfor the practice of this invention.

Identification of these epitopes will enable the analysis andquantitation of vaccinia virus-specific CD8⁺ T cells in the acute andmemory phases and to compare CD8⁺ T cell responses specific to differentepitopes. Additionally, expansion and subsequent shrinkage ofepitope-specific CD8⁺ T cells at the T cell receptor level can bemonitored. Definition of T cell epitopes will help us to betterunderstand human T cell responses to vaccinia virus as a model of humaninfection. In addition, it will provide a quantitative measure ofpoxvirus T cell immunity when these viruses are used as viral vectors.

The present invention also relates to a method of identifying thepresence of vaccinia, variola virus and/or other related poxvirus in asample comprising determining whether T cells present in the samplebecome activated in the presence of a polypeptide selected from thegroup consisting of: peptide 74A (SEQ ID NO: 1), peptide 165 (SEQ ID NO:2), an immunogenic mutant and fragment thereof and a combinationthereof, wherein if the T cells become activated, then vaccinia, variolaand/or other related poxvirus is present in the sample. In a particularembodiment, the T cells are CD8⁺ T cells.

As used herein a “sample” for use in the methods of the presentinvention can be any type of sample that can be analyzed in the methodand can be obtained from a variety of sources. T cells can be present inthe original sample or can be added to the sample. The sample can be onewhich is found in any environment, such as an unknown powder or liquid.In addition, the sample can be obtained from a host, such as a mammalianhost or individual (e.g., human, canine, feline, bovine, murine).Samples from a host include blood (e.g., whole blood, PMBCs), lymph(e.g., lymph fluid) and tissue (e.g., lymph nodes, spleen). In aparticular embodiment, the sample is from an individual that is positivefor the HLA-A0201 gene.

For example, the sample can be a sample which does not initially containT cells. In this embodiment, the sample is contacted with T cells thatbecome activated in the presence of a vaccinia, variola and/or otherrelated poxvirus. Then whether the T cells become activated in thepresence of the polypeptide is determined, wherein if the T cells becomeactivated, then vaccinia, variola and/or other related poxvirus ispresent in the sample. In another embodiment, the sample can be bloodwhich contains T cells. In this embodiment, whether the T cells becomeactivated in the presence of the polypeptide is determined, wherein ifthe T cells become activated, then vaccinia, variola and/or otherrelated poxvirus is present in the sample.

Thus, the present invention also relates to a method of determiningwhether an individual has been infected with vaccinia, variola virusand/or other related pox virus comprising determining whether theindividual's T cells become activated in the presence of polypeptideselected from the group consisting of: peptide 74A (SEQ ID NO: 1),peptide 165 (SEQ ID NO: 2), an immunogenic mutant or fragment thereofand a combination thereof, and wherein if the individual's T cellsbecome activated in the presence of the peptide, then the individual hasbeen infected with vaccinia, variola and/or other related poxvirus.

As described herein, peripheral T cells in the blood and organs of theimmune system (e.g. spleen and lymph nodes) exist in a quiescent orresting state. Upon interaction of T cells with an MHC/epitope complex,the T cells proliferate and differentiate into activated cells having avariety of functions. CD8⁺ T cells typically become cytotoxic uponactivation and destroy antigen-presenting cells via direct contact.Activated CD4⁺ T cells provide a helper function to B cells, enabling Bcells to differentiate into antibody-producing cells. Activated CD8⁺ Tcells and CD4⁺ T cells release a variety of cytokines (lymphokines orinterleukins), which can, for example, control differentiation of manyclasses of lympholytic precursor cells.

Whether the T cells present in the sample become activated can bedetermined using a variety of assays known to those of skill in the art.For example, a cytokine assay (e.g., ELISPOT), a flow cytometry assay(e.g., tetramer staining assay), intracellular cytokine staining assay(ICS) and/or a limiting dilution assay (LDA) can be used in the methodsof the present invention.

Poxviruses such as vaccinia virus allow for simplified integration ofmultiple foreign genes with high levels of expression, and thus, arewidely used for the cytoplasmic expression of recombinant genes inmammalian cells. Vaccinia virus mutants and other poxviruses arereceiving special attention because of their diminished cytopathiceffects and increased safety. For example, replicating andnon-replicating vectors encoding the bacteriophage T7 RNA polymerase fortranscription of recombinant genes and numerous cancer antigens havebeen engineered (Carroll, M. W. and Moss B., Curr. Opin. Biotechnol.,8(5):573–577 (1997); Carroll, M. W., et al., Vaccine, 15(4):387–394(1997).

The invention also relates to a method of monitoring the effectivenessof a vaccinia, variola and/or other related pox virus vaccine in anindividual who has been administered the vaccinia vaccine. The methodcomprises determining whether the individual's T cells become activatedin the presence of a polypeptide selected from the group consisting of:peptide 74A (SEQ ID NO: 1), peptide 165 (SEQ ID NO: 2), an immunogenicmutant or fragment thereof and a combination thereof, wherein if theindividual's T cells become activated, then the virus is effective inthe individual. In one embodiment, the vaccine is a vaccinia vaccine. Inanother embodiment, the vaccine is vaccinia virus that is a cancervaccine.

Exemplification

Donors

Donors in this study were three HLA-A0201-positive laboratory workersreceived primary immunization by scarification with the licensedsmallpox vaccine, Dryvax®, as recommended by the Centers for DiseaseControl and Prevention for laboratory personnel working with vacciniaviruses. The HLA-A and B alleles of donor 1 were A2 (A0201), B15, B18;those of donor 2 were A2 (A0201), B15, B44; and those of donor 3 were A2(A0201), A31, B40, B51.

Viruses

Vaccinia virus New York City Board of Health (NYCBH), the same strainused to produce Dryvaxâ, was provided by Gail Mazzara and DennisPanicali of Applied Biotechnology, Inc, and propagated and titrated inCV-1 cells (ATCC #CCL-70) as previously described (Littuau, R. A., etal., J. Virol., 66:2274–2280 (1992); Terajima, M. et al., Virus Res.84:67–77 (2002)). Modified vaccinia virus Ankara strain (MVA) was kindlysupplied by Bernard Moss of National Institute of Allergy and InfectiousDiseases/National Institute of Health, and was propagated and titratedin BHK-21 cells (ATCC #CCL-10) following published methods (Carroll, M.W., et al., Virology 238:198–211 (1997)).

CTL Lines

Vaccinia virus-specific CTL lines were isolated from peripheral bloodmononuclear cells (PBMCs) of immunized donors by limiting dilutioncloning (Demkowicz, W. E., et al., J. Virol. 67:1538–1544 (1993)).Vaccinia virus NYCBH strain was used to stimulate PBMCs for cloning andto infect target cells for cytotoxicity assays. Cytotoxicity assays wereperformed as previously described (Frey, S. E., et al., J. Med.346:1275–1280 (2002)). Hmy C1R A2.1 cells (gift from William E. Biddisonof NIH/NINDS), which express only HLA-A0201 at normal levels, were usedas targets in cytotoxicity assay to confirm the HLA-A0201 restriction.Surface expression of CD4 and CD8 was determined by flow cytometry usingFITC-conjugated antibodies (Becton Dickinson). Cross-reactivity of CTLlines was determined using autologous B-LCLs (B-lymphoblastoid celllines), that were infected with MVA as target cells in cytotoxicityassays.

Screening Peptides in Cytotoxicity Assay

Peptides were synthesized with a Symphony automated peptide synthesizerat the Protein Core Facility in the University of Massachusetts MedicalSchool or purchased from Mimotopes Pty. Ltd. When predicted epitopesoverlapped, we made them as a longer peptide fragment. For technicalreasons some of these screening peptides were made as a 13mer instead ofa 9mer. In screening cytotoxicity assays, mixtures of five peptides wereused and the concentration of each peptide was 5 mg/ml. When the peptiderecognized was longer than 9 amino acids, truncated 9mer peptideepitopes were constructed and analyzed in cytotoxicity assays. Allpeptides recognized were tested in dose-response experiments (FIG. 1).

Tetramer Staining

Peptide/HLA-A0201 tetramers were made in the Tetramer Core Facility inthe University of Massachusetts Medical School following the protocolpublished previously (Catalina, M. D., et al., J. Immunol.,167:4450–4457 (2001)). Each lot of tetramer was titrated using CTL linesspecific to the peptide mixed with autologous PBMCs at a 1 to 10 (or 20)ratio to determine the optimal concentration for staining.

Interferon (IFN)-g ELISPOT Assay

IFN-g ELISPOT assays were performed as previously described (Ennis, F.,et al., J. Infect. Dis. 185:1657–1659 (2002)). For stimulation, PBMCswere incubated with vaccinia virus NYCBH strain at an MOI of 1 or withpeptide at a final concentration of 10 mg/ml for 16 hours.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of identifying T cells in a sample that become activated inthe presence of a vaccinia or variola virus that comprises a polypeptidehaving an amino acid sequence that is identical or substantiallyhomologous to peptide 165 (SEQ ID NO: 2), comprising contacting the Tcells with a polypeptide selected from the group consisting of: a)peptide 165 (SEQ ID NO: 2), b) an immunogenic mutant or fragment of SEQID NO: 2, wherein the immunogenic mutant or fragment maintains thefunction of peptide 165 as a CD8 T cell epitope of the vaccinia orvariola virus, and c) a combination thereof, wherein activation of the Tcells by the polypeptide indicates that the T cells become activated inthe presence of the vaccinia or variola virus.
 2. The method of claim 1wherein whether the T cells present in the sample become activated isdetermined using an assay selected from the group consisting of: acytokine assay, a flow cytometry assay and a limiting dilution assay. 3.The method of claim 2 wherein the cytokine assay is an ELISPOT assay andthe flow cytometry assay is a tetramer staining assay.
 4. The method ofclaim 1 wherein the sample is selected from the group consisting of:blood, lymph and tissue.
 5. The method of claim 4 wherein the sample isa peripheral blood mononuclear cell sample.
 6. A method of determiningwhether an individual has been infected with a vaccinia or variola virusthat comprises a polypeptide having an amino acid sequence that isidentical or substantially homologous to peptide 165 (SEQ ID NO: 2),comprising determining whether the individual's T cells become activatedin the presence of a polypeptide selected from the group consisting of:peptide 165 (SEQ ID NO: 2), an immunogenic mutant or fragment of SEQ IDNO: 2, wherein the immunogenic mutant or fragment maintains the functionof peptide 165 as a CD8 T cell epitope of the vaccinia or variola virus,and a combination thereof, wherein if the individual's T cells becomeactivated in the presence of the peptide, then the individual has beeninfected with the vaccinia or variola virus.
 7. The method of claim 6wherein the individual's T cells are present in a sample, and the sampleis selected from the group consisting of: blood, lymph and tissue. 8.The method of claim 7 wherein the sample is a peripheral bloodmononuclear cell sample.
 9. The method of claim 6 wherein whether thewhether the individual's T cells become activated is determined using anassay selected from the group consisting of: a cytokine assay, a flowcytometry assay and a limiting dilution assay.
 10. The method of claim 9wherein the cytokine assay is an ELISPOT assay and the flow cytometryassay is a tetramer staining assay.
 11. A method of monitoring theeffectiveness of a vaccinia vaccine that comprises a polypeptide havingan amino acid sequence that is identical or substantially homologous topeptide 165 (SEQ ID NO: 2) in an individual who has been administeredthe vaccinia vaccine, comprising determining whether the individual's Tcells become activated in the presence of a polypeptide selected fromthe group consisting of: peptide 165 (SEQ ID NO: 2), an immunogenicmutant or fragment of SEQ ID NO: 2, wherein the immunogenic mutant orfragment maintains the function of peptide 165 as a CD8 T cell epitopeof the vaccinia or variola virus, and a combination thereof, wherein ifthe individual's T cells become activated, then the vaccinia vaccine iseffective in the individual.
 12. The method of claim 11 wherein theindividual's T cells are present in a sample, and the sample is selectedfrom the group consisting of: blood, lymph and tissue.
 13. The method ofclaim 12 wherein the sample is a peripheral blood mononuclear cellsample.
 14. The method of claim 11 wherein whether the whether theindividual's T cells become activated is determined using an assayselected from the group consisting of: a cytokine assay, a flowcytometry assay and a limiting dilution assay.
 15. The method of claim14 wherein the cytokine assay is an ELISPOT assay and the flow cytometryassay is a tetramer staining assay.
 16. The method of claim 11 whereinthe vaccinia vaccine is a cancer vaccine.